Thermal unfolding and refolding of protein under osmotic pressure clarified by wide-angle X-ray scattering

► We clarified a protein structure under osmotic pressure by wide-angle X-ray scattering. ► The osmotic pressure was varied by adding high-molecular weight co-solutes. ► Crowding by co-solutes induced a new stable and compact structure of protein. ► Hydration shell density of the protein was changed. ► In addition, the unfolding intermediate in thermal transition was stabilized by co-solutes.